Determination of a specific immunoglobulin using multiple antigens

ABSTRACT

The invention concerns a method for the immunological determination of a specific antibody in a sample liquid in which the sample liquid is incubated in the presence of a solid phase with two antigens directed against the antibody to be determined of which the first antigen carries at least one marker group and the second antigen is (a) bound to the solid phase or (b) is present in a form capable of binding to the solid phase and the antibody to be determined is detected by determining the marker group in the solid phase or/and in the liquid phase characterized in that at least one of the two antigens contains several epitope regions which react with the antibody to be determined.

The present invention concerns a method for the determination of aspecific immunoglobulin using antigens that comprise several epitoperegions.

The detection of immunoglobulins in body fluids, in particular in humansera, is used to diagnose infections with microorganisms, in particularviruses, such as HIV, hepatitis viruses etc. The presence of specificimmunoglobulins in the examined sample is usually detected by reactionwith one or several antigens that react with the specificimmunoglobulins. Methods for the determination of specificimmunoglobulins in the sample liquid must be sensitive, reliable, simpleand rapid.

In recent years more and more detection systems based on non-radioactivemarker groups have been developed in which the presence of an analyte,e.g. a specific antibody, in the examined sample can be determined withthe aid of optical (e.g. luminescence or fluorescence), NMR-active ormetal-precipitating detection systems.

EP-A-0 307 149 discloses an immunological test for an antibody in whichtwo recombinant polypeptides are used as antigens one of which isimmobilized on a solid phase and the other carries a marker group andboth recombinant antigens are expressed in different organisms toincrease the specificity of the test.

EP-A-0 366 673 discloses a method for the detection of antibodies in asample in which an antibody is detected by reaction with a purifiedlabelled antigen and with the same purified antigen in a solidphase-bound form. Human IgG is for example disclosed as an antigen.

EP-A-0 386 713 describes a method for the detection of antibodiesagainst HIV using two solid supports in which various HIV antigens areimmobilized on the two solid supports each of which is brought intocontact with an aliquot of a sample and with a labelled HIV antigenwherein the presence of antibodies is detected by a positive reaction inat least one of the tests. Recombinantly produced polypeptides aredisclosed as HIV antigens.

EP-A-0 507 586 describes a method for carrying out an immunological testfor a specific immunoglobulin in which a sample is brought into contactwith two antigens capable of binding the immunoglobulin, wherein thefirst antigen carries a group suitable for binding to a solid supportand the second antigen carries a marker group. The marker group can be adirect marker group e.g. an enzyme, a chromogen, a metal particle, oralso an indirect marker group i.e. the marker group attached to theantigen can react with a receptor for the marker group which in turncarries a signal-generating group. A fluorescein derivative is mentionedas an example of such an indirect marker group, the receptor of which isan antibody which in turn is coupled to an enzyme. Polypeptides such asthe hepatitis B surface antigen are disclosed as antigens. SH groups areintroduced into this antigen by derivatization which are used to couplethe fluorescein.

EP-A-0 507 587 discloses a specific method for the detection of IgMantibodies in which the sample is incubated with a labelled antigenwhich is directed against the antibody to be detected and with a secondantibody which is also directed against the antibody to be detected andis capable of binding to a solid phase.

However, the immunological methods of detection according to the bridgetest concept which are known from the state of the art in which alabelled antigen and an antigen capable of binding to a solid phase areused, still have major weaknesses. In particular they have a lowsensitivity when a relatively low affinity is present between theantibody to be determined and the antigen. This is especially the casefor a seroconversion that has occurred only recently and/or when newsubtypes of the infectious microorganism occur. A further disadvantageof the previously known bridge test concepts is the risk of a falsenegative evaluation of high-titre samples due to the Hook effect.

The object of the present invention was therefore to provide a methodfor the detection of specific antibodies in which the disadvantages ofthe state of the art are at least partially eliminated and which has anadequate sensitivity especially in the case of a seroconversion whichhas only recently occurred and in the case of new microorganismsubtypes. In addition the method according to the invention is intendedto reduce the Hook effect.

This object is achieved by a method for the immunological determinationof a specific antibody in a sample liquid in which the sample liquid isincubated in the presence of a solid phase with two antigens directedagainst the antibody to be determined in which the first antigen carriesat least one marker group and the second antigen is (a) bound to thesolid phase or (b) is present in a form capable of binding to the solidphase and the antibody to be determined is detected by determining themarker group in the solid phase or/and in the liquid phase characterizedin that at least one of the two antigens comprises several epitoperegions which react with the antibody to be determined.

Surprisingly it was found in bridge test immunoassays that thesensitivity of the test, especially for sera containing antibodies whichhave a low affinity for the antigen used, is improved by using at leastone multimeric antigen i.e. an antigen with multiple epitope regions. Inaddition the method according to the invention leads to a considerablereduction of the risk of false negative evaluations of high-titresamples due to the Hook effect. The optimization of the antigenpresentation by increasing the epitope density in the bridge testconcept generally leads to an improvement in the reactivity withspecific immunoglobulins in polyclonal sera as they occur in a sampleliquid such as e.g. serum. A further advantage of the method accordingto the invention is that multimeric antigens have a considerablyimproved stability compared to monomeric antigens.

Two antigens are used in a method for the immunological determination ofa specific antibody according to the bridge test concept. In a firstpreferred embodiment of the method according to the invention amultimeric antigen is used as the labelled antigen and a monomericantigen is used as the solid phase antigen. In a second embodiment ofthe method according to the invention a multimeric antigen can be usedas the solid phase antigen and a monomeric antigen can be used as thelabelled antigen. In a third preferred embodiment multimeric antigenscan be used as the labelled antigen and as the solid phase antigen.

The multimeric antigens contain multiple epitope regions i.e.structures, preferably peptide or polypeptide sequences, that reactimmunologically with the antibody to be determined. The epitope regionsare preferably linked together via immunologically inactive regions e.g.via spacer regions. Multimeric antigens are preferably used whichcomprise several identical epitope regions.

The multimeric antigens according to the invention preferably containmore than 1 to 80 immunologically reactive epitope regions andparticularly preferably more than 1 to 40 epitope regions. The epitoperegions can be coupled to a high molecular carrier or linked togetherdirectly or via spacer regions.

The epitope regions are preferably immunologically reactive syntheticpeptide sequences having a length of 6 to 50 amino acids or recombinantpolypeptide sequences having a length of preferably up to 1000 aminoacids. In addition to the actual epitope regions synthetic peptideepitopes preferably also contain a spacer region which for example canbe used for coupling to other epitopes or to a carrier or/and forcoupling marker groups or solid phase binding groups.

The spacer region is preferably an immunologically inactive peptidesequence having a length of 1 to 10 amino acids. The amino acids of thespacer region are preferably selected from the group comprising glycine,β-alanine, γ-aminobutyric acid, ε-aminocaproic acid, lysine andcompounds of the structural formula NH₂[(CH₂)_(n)0]_(x)—CH₂—CH₂—C00H inwhich n is 2 or 3 and x equals 1 to 10. The spacer region is preferablya continuous sequence of amino acids at the amino terminus or/andcarboxy terminus of the epitope region.

In an immunoassay according to the bridge test concept a first labelledantigen is used. All marker groups can be used for the method accordingto the invention e.g. radioactive and non-radioactive marker groups. Thepreferred non-radioactive marker groups can be directly or/andindirectly detectable. In the case of a directly detectable label thegroup generating a detectable measuring signal is located directly onthe antigen. Examples of such direct signal-generating groups arechromogens (fluorescent or luminescent groups, dyes), enzymes,NMR-active groups or metal particles which are coupled in a known mannerto a peptide or polypeptide antigen. The directly detectable markergroup is preferably a metal chelate detectable by fluorescence orelectrochemoluminescence and particularly preferably a rutheniumchelate, rhenium chelate, iridium chelate or osmium chelate, especiallya ruthenium chelate, e.g. a ruthenium-(bis-pyridyl)₃ ²⁺ chelate. Othersuitable metal chelate marker groups are for example described in EP-A-0580 979, WO 90/05301, WO 90/11511 and WO 92/14138. Reference is herebymade to these documents.

A further type of labelling which is suitable for the antigens accordingto the invention is an indirectly detectable label. In this type oflabelling the antigen is coupled with an indirectly detectable groupe.g. a biotin or hapten group which in turn can be detected by reactionwith a suitable binding partner (streptavidin, avidin, or anti-haptenantibody) which in turn carries a signal-generating group. An organicmolecule with a molecular weight of 100 to 2000 preferably of 150 to1000 is preferably used as an indirect marker group in the form of ahapten.

The haptens are capable of binding to a specific receptor for therespective hapten. Examples of receptors are antibodies, antibodyfragments that are directed against the hapten or another specificbinding partner for the hapten such as e.g. streptavidin or avidin ifthe hapten is biotin. The hapten is preferably selected from the groupcomprising sterols, bile acids, sexual hormones, corticoids,cardenolides, cardenolide-glycosides, bufadienolides,steroid-sapogenines and steroid alkaloids. The hapten is particularlypreferably selected from the group comprising cardenolides andcardenolide-glycosides. Representatives of these substance classes aredigoxigenin, digitoxigenin, gitoxigenin, strophanthidin, digoxin,digitoxin, ditoxin and strophanthin, digoxigenin and digoxin beingparticularly preferred. Another suitable hapten is for examplefluorescein or a suitable fluorescein derivative.

The receptor for the hapten is coupled to a signal-generating group,preferably to an enzyme such as peroxidase, alkaline phosphatase,β-galactosidase, urease or Q-β-replicase. However, the signal-generatinggroup can also be a chromogenic, radioactive or NMR-active group or ametal particle (e.g. gold). The hapten can for example be coupled to theantigen by coupling the hapten in the form of an active ester derivativeto the amino terminus or/and to free amino side groups of the peptide orpolypeptide antigen.

The term “active ester” within the sense of the present inventionencompasses activated ester groups that can react with free amino groupsof peptides under such conditions that no interfering side reactionswith other reactive groups of the peptide can occur. AnN-hydroxy-succinimide ester is preferably used as the active esterderivative. Examples of suitable hapten-active ester derivatives aredigoxin-4′″-hemiglutarate-N-hydroxy-succinimide ester,digoxigenin-3-carboxymethyl ether-N-hydroxysuccinimide ester,digoxigenin-3-O-methyl-carbonyl-ε-aminocaproic acid-N-hydroxysuccinimideester, digoxigenin-3-hemisuccinate-N-hydroxysuccinimide ester,digitoxin-4′″-hemiglutarate-N-hydroxysuccinimide ester anddigitoxigenin-3-hemisuccinate-N-hydroxysuccinimide ester. These haptenderivatives are commercially available from the Boehringer MannheimCompany GmbH (Mannheim, GER). In addition to the N-hydroxysuccinimideesters it is also possible to use analogous p-nitro-phenyl,pentafluorophenyl, imidazolyl or N-hydroxybenzo-triazolyl esters.

In addition to the first labelled antigen a second antigen is also usedin the method according to the invention which is bound to a solid phaseor is present in a form capable of binding to a solid phase and can alsobe a multimeric antigen. Binding between the solid phase antigen and thesolid phase can be covalent or adsorptive and occur directly, viachemical linker groups or via a specific interaction e.g.biotin-streptavidin/avidin, antigen-antibody, carbohydrate-lectin. Thesolid phase antigen is preferably a biotinylated antigen and the solidphase is correspondingly coated with streptavidin or avidin. Biotingroups can be coupled to the antigen in a known manner e.g. byintroduction of biotin active ester derivatives. Such methods are knownto a person skilled in the art.

The number of marker or solid phase binding groups on the multimericantigen is variable i.e. one or several groups may be present. In someembodiments of the method according to the invention it is preferable ifat least 3 and particularly preferably 3 to 20 marker or solid phasebinding groups are present. In this manner it is possible to achieve asurprisingly high improvement in sensitivity and a significant decreasein the Hook effect (false negative evaluation of strongly positivesamples).

The present invention is based on the finding that in an immunologicaltest for the determination of a specific antibody in a sample liquid itis advantageous if at least one of the two antigens used for the test isa multimeric antigen i.e. comprises several epitope regions preferablyseveral identical epitope regions. The term “epitope region” in thesense of the present invention denotes a structure, preferably a peptideor polypeptide sequence, which exhibits a specific reaction with theantibody to be determined. There are several possibilities of arrangingseveral epitope regions on the multiple antigen.

In a first embodiment a carrier which does not react with the antibodyto be determined to which the epitope regions are covalently coupled isused as the muitimeric antigen. Examples of suitable carriers arepeptides, polypeptides or synthetic carriers e.g. dextrans. Examples ofsuitable polypeptides are albumins, e.g. bovine serum albumin,unspecific immunoglobulins, immunoglobulin fragments, β-galactosidaseand polylysine. If a carrier is used care must be taken that it exhibitsno cross-reactivity with antibodies in the sample liquid.

The epitope regions are preferably coupled via a bifunctional linker toreactive groups of the carrier e.g. NH₂ groups or SH groups. Thecoupling is preferably achieved via NH₂ groups of the carrier.

In this embodiment of the invention an antigen of the general formula(P—)_(n)T(-L)_(m)  (Ia)orT(—P-L_(m))_(n)  (Ib)is preferably used in which T denotes a carrier, P denotes peptide orpolypeptide sequences which contain identical or differentimmunologically reactive epitope regions and are covalently coupled tothe carrier and L denotes marker groups or groups capable of binding toa solid phase which are covalently coupled to the carrier or to thepeptide or peptide sequences, n is a number larger than 1 to 40 and m isa number between 1 and 10. The symbols n and m do not have to denoteintegers since the coverage of the carrier with epitope groups or withmarker or solid phase binding groups can be statistical in a reactionmixture. n is preferably larger than or equal to 2.

The peptide or polypeptide sequences coupled to the carrier preferablycontain synthetic peptide sequences with a length of 6 to 50 amino acidsor recombinant polypeptide sequences with a length of preferably up to1000 amino acids.

Synthetic peptide sequences can in addition to the actual epitope regionalso optionally contain a spacer region as defined above which can forexample be located between the epitope and carrier or/and between theepitope and marker or solid phase binding group.

The peptide or polypeptide epitopes can be coupled to the carrier viathe N-terminus, the C-terminus or via reactive groups in the side chain.One method of coupling is to activate an NH₂ group of the carriermolecule by reaction with known linker substances (e.g.maleinimidohexanoic acid, maleinimidopropionic acid, maleinimidobenzoicacid) and covalently couple an SH-activated peptide derivative to thecarrier. The marker or solid phase binding groups are usually coupled tothe carrier molecule or/and to the epitope regions in the form of activeesters. However, other coupling methods are also conceivable e.g. viabifunctional photolinkers.

In order to synthesize multimeric antigens which contain the epitopescoupled to an inert carrier, the appropriate peptides are preferablysynthesised with a reactive mercapto group e.g. by introducing anadditional cysteine residue. In this case the peptide can be modifiedwith a linker either N-terminally, C-terminally or also at any positionin the sequence. For the reaction to form the multimeric antigen acarrier which contains primary amino groups can for example firstly beloaded with the appropriate active ester derivative of the marker groupand subsequently with maleinimidoalkyl groups. In this manner the aminogroups of the ε-amino side chain of lysine residues in the carrier arepartially labelled with the marker group (e.g. digoxigenin orbipyridylruthenium) or the solid phase binding group (e.g. biotin) andthe other portion is converted into maleinimide groups.

In a further step the peptide or the peptide mixture containing thedesired epitope regions is then coupled to the maleinimide-modifiedcarrier via the reactive mercapto function. If the marker group islocated directly on the peptide, the multimeric antigen is synthesizedin an analogous manner except that now an appropriately labelledSH-activated peptide is reacted with the carrier.

In a further embodiment of the invention a multimeric antigen can beused which contains several epitope regions which are covalently coupledtogether either directly or via spacer regions. The linking of theepitopes is preferably achieved at least partially via trifunctionallinker molecules so that the antigen contains at least one branchingsite and preferably 1 to 7 branching sites.

In this embodiment an antigen of the general formula II is preferablyused:P¹{P²[P³(P⁴)_(t)]_(s)}_(r)  (II)in which P¹, P², P³ and P⁴ denote peptide sequences with a length of upto 50 amino acids in which at least 2 peptide sequences containidentical or different immunologically reactive epitope regions, r is 1or 2, s is an integer from 0 to 4 and t is an integer from 0 to 8wherein the antigen contains at least one branching site and at leastone marker group or a group capable of binding to a solid phase.

The antigen of formula II forms a tree-like structure with a maximum of7 branching sites if P¹, P², P³ and P⁴ are linear peptide sequences andpreferably contains two to eight identical or different immunologicallyreactive epitope regions. The epitope regions are preferably not linkeddirectly together but via spacer regions. The spacer regions arepreferably immunologically inactive peptide sequences with a length of 1to 10 amino acids as defined above. Not all peptide sequences P¹, P², P³and P⁴ have to contain epitope regions, instead structures are alsopossible in which these sequences only consist of spacer regions.Branches can be incorporated into the structure by using trifunctionalamino acids e.g. lysine or ornithine.

In addition the antigen of the general formula II contains at least onemarker or solid phase binding group as defined above. These groups canfor example be coupled selectively to the ends or/and to reactive sidechains of the peptide sequences.

So-called mosaic proteins are yet a further embodiment of multimericantigens i.e. recombinant fusion polypeptides whose amino acid sequencecontains several immunologically reactive epitope regions which areoptionally linked via immunologically inactive spacer regions. Therecombinant mosaic proteins are obtainable by synthesizing a DNAsequence coding for the desired protein and expressing it in arecombinant host cell. Such procedures are known to a person skilled inthe area of molecular biology and are described in standard textbooks(e.g. Sambrook et al., Molecular Cloning. A Laboratory Manual, 2ndEdition (1989), Cold Spring Harbor Laboratory Press). Marker or solidphase binding groups can also be introduced into the recombinant proteinaccording to known methods.

In a further preferred embodiment of the invention the epitope regionsare synthetic peptide sequences with a length of 6 to a maximum of 50particularly preferably up to a maximum of 30 amino acids. Marker groupsor solid phase binding groups can be selectively introduced into suchepitope regions with regard to their location as well as with regard totheir number. Thus in the synthetic production by using certainprotecting groups on reactive side groups e.g. primary amino groups ofthe amino acid derivatives used it is possible to specifically selectthose positions of the peptide which are available for reaction with theintroduced marker group after selective cleavage of the protectinggroup.

For this the peptide having the desired amino acid sequence issynthesized on a solid phase preferably using a commercial peptidesynthesizer (e.g. the instruments A 431 or A 433 from AppliedBiosystems). The synthesis is carried out according to known methodspreferably starting at the carboxyl terminus of the peptide using aminoacid derivatives. Amino acid derivatives are preferably used whose aminoterminal groups required for coupling are derivatized with afluorenylmethyloxycarbonyl (Fmoc) residue. Reactive side groups of theamino acids used contain protecting groups that can be readily cleavedoff after completion of the peptide synthesis. Preferred examples ofthis are protecting groups such as triphenylmethyl (Trt), t-butyl ether(tBu), t-butyl ester (0 tBu), tert.-butoxycarbonyl (Boc) or2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc).

The amino side chains of lysine residues or of other amino acidderivatives with primary amino side groups that are located at positionsof the peptide which are later intended to be derivatized with thehapten are provided with a first amino protecting group which isselected such that it can be quantitatively cleaved off under particularreaction conditions e.g. in the presence of acid. An example of asuitable acid-labile protecting group is Boc. The side groups of lysineresidues or of other amino acid residues with primary amino side groupsto which no coupling of a hapten is desired are provided with a secondamino-protecting group which is selected such that it cannot itself becleaved off under conditions under which the first protecting group canbe cleaved off. The second protecting group is also preferably stableunder those conditions under which the peptide is cleaved from the solidphase and under which all other protecting groups are cleaved off.Examples of such second protecting groups are acid-resistant protectinggroups such as phenylacetyl. In addition to the 20 natural amino acidsthe peptide can also contain artificial amino acids such as β-alanine,γ-amino-butyric acid, ε-amino-caproic acid or norleucine. Theseartificial amino acids are used for the synthesis in a protected formanalogously to the natural amino acids.

After completion of the synthesis protecting groups, including the firstamino-protecting groups, which are located at the positions at which thecoupling of the hapten is to take place are cleaved, optionally afterreleasing the peptide from the solid phase. Then the product obtained inthis manner is purified, preferably by HPLC. Subsequently the haptenlabel is introduced by reacting the peptide with the hapten-active esterderivative desired in each case which reacts with free primary aminogroups i.e. with the amino terminal group or/and amino side groups ofthe peptide. Preferably 1.5 to 2.5 equivalents of active ester are usedper free primary amino group. Subsequently the reaction product ispurified, preferably by HPLC.

If the peptide still contains amino groups that are derivatized with asecond protecting group such as phenylacetyl then these protectinggroups are removed in the last step. Phenylacetyl protecting groups canfor example be enzymatically removed at room temperature withimmobilized or soluble penicillin G amidase in aqueous solutioncontaining an organic solvent.

If the peptides produced by the process according to the inventioncontain an intramolecular disulfide bridge, then the peptide sequencecan be oxidized on the solid phase with for example iodine inhexafluoroisopropanol/dichloromethane (Cober et al. The Peptide,Academic Press, New York, 1981, pages 145 to 147) after completion ofthe synthesis but before cleaving the N-terminal Fmoc-protecting groupof the last amino acid, and subsequently the N-terminal Fmoc-protectinggroup is cleaved.

A reactive SH group can for example be introduced by coupling a cysteineresidue to the amino terminus of the peptide.

Metal chelate marker groups are introduced into synthetic peptides (a)after synthesis of the desired peptide sequence and preferably beforecleavage of the peptide from the solid phase and before cleavage ofprotecting groups to reactive side groups of the amino acid derivativesused for the peptide synthesis by coupling an activated luminescentmetal chelate e.g. an active ester derivative to the N-terminal primaryamino group of the peptide and/or (b) during the synthesis of thepeptide by introducing amino acid derivatives which are coupledcovalently to a luminescent metal chelate marker group e.g. by means ofa ε-derivatized lysine.

Branched multimeric antigens can be synthesized by using adiaminocarboxylic acid such as lysine protected by two Fmoc groups. Thepeptides can for example be biotinylated by introducing a biotinderivative at the N-terminus while the peptide is still coupled to thesolid phase.

Peptide epitopes or polypeptide epitopes from pathogenic organisms e.g.bacteria, viruses and protozoa or from autoimmune antigens arepreferably used for the method according to the invention. Theimmunologically reactive epitope region is preferably derived from viralantigens e.g. the amino acid sequences of HIV I, HIV II, HIV subtype Oor hepatitis C-virus (HCV).

Preferably HIV I or HIV II or subtype 0 epitopes are selected from theregions gp32, gp41 gp120 and gp24. HCV epitopes are preferably selectedfrom the Core/Env region or the non-structural protein regions NS3, NS4or NS5.

The epitope region of HIV I or HIV II or HIV subtype 0 amino acidsequences is particularly preferably selected from the group of aminoacid sequences: NNTRKSISIG PGRAFYT (I) NTTRSISIGP GRAFYT (II) IDIQEERRMRIGPGMAWYS (III) QARILAVERY LKDQQLLGIW GASG (IV) LGIWGCSGKL ICTTAVPWNASWS (V) KDQQLLGIWG SSGKL (VI) ALETLLQNQQ LLSLW (VII) LSLWGCKGKL VCYTS(VIII) WGIRQLRARL LALETLLQN (IX) and QAQLNSWGCA FRQVCHTTVP WPNDSLT (X)or partial sequences thereof which have a length of at least 6 andpreferably of at least 8 amino acids.

The amino acid sequences I to III are derived from the gp120 region ofHIV I, the amino acid sequences IV to IX are derived from the gp41region of HIV I and the amino acid sequence X is derived from the gp32region of HIV II. The amino acid sequences I to X are also shown in thesequence protocols SEQ ID NO. 1 to SEQ ID NO. 10. Each of the sequencesV, VIII and X contain two cysteine residues which are preferably presentin the form of a disulfide bridge.

The epitope region of HCV amino acid sequences is preferably selectedfrom the group of the amino acid sequences: SRRFAQALPV WARPD (XI)PQDVKFPGGG QIVGGV (XII) EEASQHLPYI EQ (XIII) QKALGLLQT (XIV) SRGNHVSPTHYVPESDAA (XV) PQRKNKRNTN RRPQDVKFPG GGQIVGVV (XVI) and AWYELTPAETTVRLRAYNNT PGLPV (XVII)or partial sequences thereof which have a length of at least 6 andpreferably at least 8 amino acids. The sequence XI is derived from theNS5 region, the sequences XII and XVI from the Core region, thesequences XIII, XIV and XV from the NS4 region and the sequence XVII isderived from the NS3 region of HCV. The amino acid sequences XI to XVIIare also shown in the sequence protocols SEQ ID NO. 11 to SEQ ID NO. 17.

The present invention in addition concerns a reagent for theimmunological determination of a specific antibody in a sample liquidcomprising a reactive phase, two antigens directed against the antibodyto be determined of which the first antigen carries a marker group andthe second antigen is (a) bound to the solid phase or (b) is present ina form capable of binding to the solid phase characterized in that atleast one of the two antigens contains several epitope regions whichreact to the antibody to be determined.

In one embodiment of the present invention the reagent contains a firstlabelled antigen with several epitope regions which carries at least onehapten marker group and a receptor for the hapten which in turn containsa signal-generating group. In addition a reagent is preferred whichcomprises a second solid phase antigen with several epitope regionswhich carries at least one biotin group and a reactive solid phasecoated with streptavidin or avidin.

Yet a further subject matter of the present invention is the use ofmultimeric antigens which contains several immunologically reactiveepitope regions in an immunological test procedure to determine specificantibodies in a sample liquid.

Those antibodies are preferably determined which indicate an infectionby microorganisms such as bacteria, viruses or protozoa. Antibodiesdirected against viruses such as e.g. against HIV or hepatitis virusesare particularly preferably determined. The sample liquid is preferablyserum, particularly preferably human serum. In addition it is preferredthat the multimeric antigens according to the invention are used in animmunological method in a bridge test format.

The test procedure preferably comprises mixing the sample liquid withthe first antigen and the second antigen on the solid phase in order toobtain a labelled immobilized complex of first antigen, antibody andsolid phase-bound second antigen. Compared to other test formats fordetecting antibodies, the bridge test format leads to an improvement insensitivity i.e. all immunoglobulin classes such as IgG, IgM, IgA andIgE are recognized as well as in specificity i.e. the unspecificreactivity is reduced. The specificity and sensitivity of the doubleantigen bridge test can be further improved if a two step test procedureis used in which the sample liquid is mixed with the first and thesecond antigen in a first step and subsequently the receptor for thehapten label of the first antigen which carries the signal-generatinggroup is added after 1 to 4 h, particularly preferably after 1.5 to 2.5h.

The invention finally also concerns new antigens of formulae (Ia), (Ib)and (II) as defined above.

The present invention is further described by the following examples,sequence protocols and figures.

SEQ ID NO. 1: shows the amino acid sequence of an epitope from the gp120region of HIV I;

SEQ ID NO. 2: shows the amino acid sequence of a further epitope fromthe gp120 region of HIV I;

SEQ ID NO. 3: shows the amino acid sequence of a further epitope fromthe gp120 region of HIV I, subtype O;

SEQ ID NO. 4: shows the amino acid sequence of an epitope from the gp41region of HIV I;

SEQ ID NO. 5: shows the amino acid sequence of a further epitope fromthe gp41 region of HIV I;

SEQ ID NO. 6: shows the amino acid sequence of yet a further epitopefrom the gp41 region of HIV I;

SEQ ID NO. 7: shows the amino acid sequence of an epitope from the gp41region of HIV I, subtype O;

SEQ ID NO. 8: shows the amino acid sequence of a further epitope fromthe gp41 region of HIV I, subtype 0;

SEQ ID NO. 9: shows the amino acid sequence of yet a further epitopefrom the gp41 region of HIV I, subtype O;

SEQ ID NO.10: shows the amino acid sequence of an epitope from the gp32region of HIV II;

SEQ ID NO.11: shows the amino acid sequence of an epitope from the NS5region of HCV;

SEQ ID NO.12: shows the amino acid sequence of an epitope from the Coreregion of HCV;

SEQ ID NO.13: shows the amino acid sequence of an epitope from the NS4region of HCV;

SEQ ID NO.14: shows the amino acid sequence of a further epitope fromthe NS4 region of HCV;

SEQ ID NO.15: shows the amino acid sequence of yet a further epitopefrom the NS4 region of HCV;

SEQ ID NO.16: shows the amino acid sequence of a further epitope fromthe Core region of HCV and

SEQ ID NO.17: shows the amino acid sequence of an epitope from the NS3region of HCV;

FIG. 1: shows the amino acid sequence of the recombinant HIV p24antigen,

FIG. 2: shows a comparison of the measured signals in a double antigenbridge test when using a monomeric and a multimeric ruthenylatedHIV-gp120 antigen and

FIG. 3: shows a comparison of the measured signals in a double antigenbridge test when using a monomeric and a multimeric biotinylatedHIV-gp41 antigen.

EXAMPLE 1

Synthesis of Peptide Epitope Regions

The peptide epitope regions were synthesized by means offluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis on abatch peptide synthesizer e.g. from Applied Biosystems A431 or A433. Forthis 4.0 equivalents of each of the amino acid derivatives shown intable 1 were used: TABLE 1 A Fmoc-Ala-OH C Fmoc-Cys(Trt)-OH DFmoc-Asp(tBu)-OH E Fmoc-Glu(tBu)-OH F Fmoc-Phe-OH G Fmoc-Gly-OH HFmoc-His(Trt)-OH I Fmoc-Ile-OH K1 Fmoc-Lys(phenylacetyl)-OH K2Fmoc-Lys(Boc)-OH K3 Fmoc-Lys(Fmoc)-OH K4 Fmoc-Lys(BPRu)-OH L Fmoc-Leu-OHM Fmoc-Met-OH N Fmoc-Asn(Trt)-OH P Fmoc-Pro-OH Q Fmoc-Gln(Trt)-OH RFmoc-Arg(Pmc)-OH S Fmoc-Ser(tBu)-OH T Fmoc-Thr(tBu)-OH UFmoc-βAlanine-OH V Fmoc-Val-OH W Fmoc-Trp-OH Y Fmoc-Tyr(tBu)-OH ZFmoc-ε-aminocaproic acid-OH Nle Fmoc-ε-norleucine-OH AbuFmoc-γ-aminobutyric acid-OH

If cysteine residues are present in the peptide sequence, an oxidationon the solid phase is carried out immediately after completion of thesynthesis using iodine in hexafluoroisopropanol/dichloromethane.

The amino acids or amino acid derivatives were dissolved inN-methylpyrrolidone. The peptide was synthesized on 400-500 mg4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin (TetrahedronLetters 28 (1987), 2107) with a loading of 0.4-0.7 mmol/g (JACS 95(1973), 1328). The coupling reactions were carried out for 20 minutes indimethylformamide as the reaction medium with 4 equivalentsdicyclohexylcarbodiimide and 4 equivalents N-hydroxybenzotriazolrelative to the Fmoc-amino acid derivative. The Fmoc group was cleavedwithin 20 minutes after each synthesis step using 20% piperidine indimethylformamide.

The release of the peptide from the synthesis resin and the cleavage ofthe acid-labile protecting groups—with the exception of the phenylacetylprotecting group—was achieved within 40 min at room temperature with 20ml trifluoro acetic acid, 0.5 ml ethanedithiol, 1 ml thioanisol, 1.5 gphenol and 1 ml water. The reaction solution was subsequently admixedwith 300 ml cooled diisopropyl ether and kept at 0° C. for 40 min tocompletely precipitate the peptide. The precipitate was filtered, washedagain with diisopropyl ether, dissolved in a small amount of 50% aceticacid and lyophilized. The crude material obtained was purified for ca.120 min. by means of preparative HPLC on delta-PAK RP C18 material(column 50×300 mm, 100 Å, 15μ) using an appropriate gradient (eluant A:water, 0.1% trifluoro-acetic acid, eluant B: acetonitrile, 0.1%trifluoro-acetic acid). The identity of the eluted material was checkedby means of ion spray mass spectrometry.

The hapten label, e.g. a digoxigenin or digoxin label, was introduced insolution by coupling appropriate active ester derivatives e.g.digoxigenin-3-carboxy-methylether-N-hydroxysuccinimide ester (BoehringerMannheim GmbH, Mannheim, GER) to the free amino groups of the peptide.The peptide to be derivatized was dissolved in a mixture of DMSO and 0.1M potassium phosphate buffer pH 8.5. Subsequently 2 equivalents ofactive ester per free primary amino function dissolved in a small amountof DMSO was added dropwise and stirred at room temperature. The reactionwas monitored by means of analytical HPLC. The product is purified bymeans of preparative HPLC.

The lysine derivative K1 was used for positions at which no haptenlabelling was to take place. The lysine derivative K2 was used forpositions at which a hapten labelling was to take place. The lysinederivative K3 was used to couple the ε-amino group to the peptide in thespacer region.

If the peptide still contained lysines protected with phenylacetyl, thenthis protecting group was enzymatically cleaved at room temperature inthe last step using penicillin G amidase in an aqueous medium containinga proportion of organic solvent. The enzyme was filtered and the peptidewas purified by means of preparative HPLC. The identity of the elutedmaterial was checked by means of ion spray mass spectrometry.

A ruthenium marker group was introduced either N-terminally by means ofa ruthenium(bispyridyl)₃-carboxylic acid derivative (BPRu—C00H), e.g.Ru—(bispyridyl)₃ ²⁺—N-hydroxysuccinimide ester or into the sequence bymeans of an ε-derivatized lysine residue K4 (Fmoc-Lys(BPRu)OH).

A biotin label was introduced either N-terminally by derivatization on aresin (biotin active ester) or within the sequence analogously to theintroduction of a ruthenium label by means of a lysine appropriatelyε-derivatized with biotin.

Branched multimeric peptides were synthesized analogously to thesynthesis of the linear peptides. In this case a resin with a lowloading density e.g. with a loading of 0.2 mmol/g was selected as thesolid phase. A bis Fmoc-protected diamino carboxylic acid such asFmoc-Lys(Fmoc)-OH was used for the branching.

The prepared peptides are listed in Tables 2 and 3.

The peptide compounds shown in Tables 2a-2d were prepared from theregions gp120, gp41 and gp32 of HIV I and HIV II. TABLE 2a SH-activatedlinear peptides gp41/1 CUZU-WGIRQLRARLLALETLLQN gp41/2CUZU-LSLWGCKGKLVCYTS gp41/4 CUZU-ALETLLQNQLLSLW gp120CUZU-IDIQEMRIGPMAWYS

TABLE 2b Digoxigenin-labelled linear peptides gp120digoxigenin-3-cme-UZU-NNTRKSISIGPGRAFYTdigoxigenin-3-cme-UZ-NTTRSISIGPGRAFYdigoxigenin-3-cme-UZU-IDIQEERRMRIGPGMAWYS gp41/1digoxigenin-3-cme-UZU-AVERYLKDQQLLGIWdigoxigenin-3-cme-ZUZU-AVERYLKDQQLLGIW digoxigenin-3-cme-UZ-QARILAVERYLKDQQLLGIWGASG digoxigenin-3-cme-ZGGGG-QARILAVERYLKDQQLLGIWGASG digoxigenin-3-cme-UZU-WGIRQLRARLLALETLLQNgp41/2 digoxigenin-3-cme-UZU-LGIWGCSGKLICTTAVLGIWGCSGK-(cme-3-digoxigenin)-LICTTAVdigoxigenin-3-cme-UZU-LGIWGCSGK-(cme-3- digoxigenin)-LICTTAVdigoxigenin-3-cme-ZU-GCSGKLICTTAVPWNASWSGCSGK-(cme-3-digoxigenin)-LICTTAVPWNASWSGCSGKLICTTAVPWNASWSK(cme-3-digoxigenin)Gdigoxigenin-3-cme-UZU-LSLWGCKGKLVCYTS gp41/3digoxigenin-3-cme-UZU-KDQQLLGIWGSSGKL gp41/4digoxigenin-3-cme-UZU-ALETLLQNQLLSLW gp32digoxigenin-3-cme-Z-NSWGCAFRQVCHTT

TABLE 2c Ruthenylated linear peptides gp120 BPRu-UZU-NNTRKSISIGPGRAFYTBPRu-UZ-NTTRSISIGRGRAFY BPRu(ethyleneglycol)-UZ-NTTRSISIGPGRAFYPBRu-UZU-IDIQEERRMRIGPGMAWYS gp41/1 PBRu-UZU-AVERYLKDQQLLGIWBPRu-UGGG-QARILAVERYLKDQQLLGIWGASG BPRu-GGGG-QARILAVERYLKDQQLLGIWGASGBPRu-UZU-WGIRQLRARLLALETLLQN gp41/2 BPRu-UZU-LGIWGCSGKLICTTAVBPRu-UGGG-GCSGKLICTTAVPWNASWS (GCSGKLICTTAVPWNASWS)K-(BPRu) gp41/3BPRu-UZU-KDQQLLGIWGSSGKL gp41/4 BPRu-UZU-ALETLLQNALLSLW gp32BPRu-UZU-NSWGCAFRQVCHTT BPRu-GGG-QAQLNSWGCAFRQVCHTTVPWPNDSLT

TABLE 2d Branched peptides gp120 (NTTRSISIGPGRAFY-AbuZAbuZ)₂-K-Z-AbuZ-K-(Bi) ((NTTRSISIGPGRAFY-ZU)₂-K-UU-K-(Bi)((NNTRKSISIGPGRAFYT-UZU-K)₂-UZU- NNTRKSISIGPGRAFYT-UZU-K)₂-UZU-Bi gp120(NTTRSISIGPGRAFY-ZU)₂-K-UU-K-(BPRu)

The peptides shown in the following Tables 3a-d were synthesized fromthe NS5 region, the NS4 region, the Core region and the NS3 region ofHCV. TABLE 3a SH-activated linear peptides NS4/3 C-UZ-SRGNHVSPTHYVPESDAA

TABLE 3b Hapten-labelled linear peptides NS5/1digoxigenin-3-cme-UZU-SRRFAQALPVWARPD Core2mdigoxigenin-3-cme-U-PQDVKFPGGGQIVGGV NS4/1digoxigenin-3-cme-UU-Nle-EEASQHLPYIEQ NS4/2digoxigenin-3-cme-UU-QKALGLLQT NS4/3digoxigenin-3-cme-UZU-SRGNHVSPTHYVPESDAA Core1digoxigenin-3-cme-UZU-KNKRNTNRR Core1 + 2 digoxigenin-3-cme-U-PQRKNKRNTNRRPQDVKFPGGGQIVGVV NS3/1 digoxigenin-3-cme-UZ-AWYELTPAETTVRLRAYMNTPGLPV

Example 3c: Ruthenylated linear peptides Core1 BPRu-GGGG-KNKRNTNRRCore1 + 2 BPRu-UZU-KNKRNTNRRPQDVKFPGGGQIVGGV NS4/1 + 2BPRu-UZU-SQHLPYIEQG-NleNle- LAEQFKQQALGLLQT NS4/3mBPRu-UZ-SRGNHVSPTHYVPESDAA NS5/1 BPRu-UZ-SRRFAQALPVWARPD Core1 + 2 + 3BPRz-UZ- KNKRNTNRRPQDVKFPGGGQIVGGVLLPRR Core1m BPRu-UZ-NPKPQKKNKRNTNRRCore3m BPRu-UZ-GQIVGGVYLLPRRGPRLG Core2m BPRu-UZ-PQDVKFPGGGQIVGGVNS4/3m-I BPRuz-UZU-SRGNHVSPTHYVPESDAA NS4/1 BPRu-UZU-SQHLPYIEQ

TABLE 3d Branched peptides NS4/3m (SRGNHVSPTHYVPESDAA-UU)₂ KUUK (BPRu)(SRGNHVSPTHYVPESDAA-UU)₄ K₂KUUK (BPRu) (SRGNHVSPTHYVPESDAA-UU)₈K₄U₄K₂KUUK (BPRu) (SRGNHVSPTHYVPESDAA-UU)₂ KUUK (Z-Bi)(SRGNHVSPTHYVPESDAA-UU)₄ K₂KUUK (Z-Bi) (SRGNHVSPTHYVPESDAA-UU)₈ K₄U₄K₂KUUK (Z-Bi)

EXAMPLE 2

Synthesis of Carrier-Bound Multimeric Antigens (Polyhaptens) withPeptide Epitopes

The appropriate peptides were synthesized with a reactive mercaptofunction e.g. by introducing an additional cysteine (cf. Tables 2a and2b). In this process the peptide can be modified with a so-called linkereither N-, or C-terminally or at any desired position in the sequence.The corresponding peptides were synthesized as described in example 1.

For the reaction to form the polyhapten the carrier containing NH₂groups was firstly loaded with the appropriate active ester of themarker groups and subsequently with maleinimidoalkyl groups, preferablyby treatment with maleinimidohexyl-(MHS) ormaleinimidopropyl-N-hydroxysuccinimide ester (MPS). By this means theprimary amino groups in the carrier (e.g. ε-amino side chain of lysineresidues) were partially labelled and the other part was converted intomaleinimide groups.

The carrier was preferably reacted with the active esters in 0.1 mol/lpotassium phosphate buffer pH 7.0-8.5 within 2-4 h at room temperatureusing a concentration of 5-20 mg/ml. The lower molecular components wereeither separated by dialysis or gel chromatography (AcA 202-Gel, eluant0.1 mol/l potassium phosphate buffer pH 7-8.5).

The peptide or the peptide mixture was then coupled within 6 h at roomtemperature in a further step with the reactive mercapto function on theMHS-modified labelled carrier in 0.1 mol/l potassium phosphate buffer pH8.5. Non-reacted peptide was either separated by dialysis or gelchromatography.

If the label was to be located directly on the peptide, the polyhaptenwas synthesized analogously and an appropriately labelled SH-activatedpeptide was used.

Rabbit IgG, bovine serum albumin, β-galactosidase, amino-dextran andbovine Fab antibody fragments were used as carriers. The loading of thecarrier with the peptide sequences was 1:2-1:20 on a molar basis. Theloading of the carrier with marker groups was 1:1 to 1:20 on a molarbasis.

EXAMPLE 3

Synthesis of carrier-bound multimeric antigens (polyhaptens) containingpolypeptide epitopes as exemplified by poly-p24-BSA-BPRu

1. Principle

Bovine serum albumin (BSA) was reacted in the stated order withruthenium-(bis-pyridyl)₃ ²⁺-N-hydroxy-succinimide ester (BPRU) andmaleinimidohexanoyl-N-hydroxysuccinimide ester (MHS) and dialysed ineach case to separate the free, non-bound derivatization reagents.

Recombinant p24 antigen from E. coli (Ghrayeb and Chang, DNA5 (1986),93-99) with the amino acid sequence shown in FIG. 1 was reacted withN-succinimidyl-S-acetylthio propionate (SATP) to introduce thiolresidues via amino groups and dialysed to separate free non-bound SATP.

After releasing the SH groups in the activated p24 antigen it wascoupled to the maleinimido functions of BSA-BPRu. Excess functionalcoupling groups were captured with cysteine and N-methylmaleinimide andthe reaction was thus terminated.

The product was then isolated from the reaction mixture bychromatography on Sephacryl S 200.

2.1 Synthesis of BSA (MH)—BPRu

A 5-fold molar excess of BPRU reagent (0.4 ml BPRU stock solutioncontaining 47 mg/ml in DMSO) was added to 250 mg BSA at a proteinconcentration of 20 mg/ml in PBS buffer pH 8.0.

After the addition it was stirred for a further 75 min at 25° C. Thereaction was then stopped by addition of lysine to a final concentrationof 10 mmol/l and stirred for a further 30 min at 25° C.

SH groups of BSA that are present were derivatized by addition ofiodoacetamide to a final concentration of 10 mmol/l. For this purposethe mixture was stirred for a further 45 min at 25° C. and pH 8.0.

Free non-bound derivatization reagents were completely separated bydialysis (20 hours, 4° C.) against >500-fold volume of PBS buffer pH 7.5(50 mmol/l Na phosphate, 150 mmol/l NaCl, pH 7.5).

The incorporation of BPRU was 4.7 moles per mole BSA. The yield was 220mg BSA-BPRU (89%).

Then a 25-fold molar excess of MHS reagent (0.5 ml MHS stock solutioncontaining 50 mg/ml in DMSO) was added to 220 mg BSA-BPRU at a proteinconcentration of 20 mg/ml in PBS buffer pH 7.1 and stirred for a further60 min at 25° C.

The reaction was stopped by addition of lysine to a final concentrationof 10 mmol/l and stirred for a further 30 min at 25° C.

Free non-bound MHS reagent was completely separated by dialysis (20hours, 4° C.) against >500-fold volume PBS buffer pH 7.5. Yield: 210 mgBSA(MG)-BPRU (84%).

2.2 Synthesis of p24 Antigen (SATP)

A 3-fold molar excess of SATP reagent (0.06 ml SATP stock solutioncontaining 35 mg/ml in DMSO) was added to 100 mg p24 antigen at aprotein concentration of 10 mg/ml in 0.1 M Na phosphate, 0.1% (w/v) SDS,pH 7.1 and it was stirred for a further 60 min at 25° C.

The reaction was then stopped by addition of lysine to a finalconcentration of 10 mmol/l and stirred for a further 30 min at 25° C.

Free non-bound SATP reagent was subsequently completely separated bydialysis (20 hours, room temperature) against >500-fold volume 0.1 mol/lNa-phosphate, 0.1% (w/v) SDS, pH 6.5.

Yield: 95 mg p24 antigen (SATP) (95%).

2.3 Synthesis of poly-p24-Antigen BSA-BPRU

Hydroxylamine (1 mol/l; Merck) was added to a final concentration of 30mmol/l to 95 mg p24 antigen (SATP) at a protein concentration of 10mg/ml in 0.1 mol/l Na-phosphate, 0.1% (w/v) SDS, pH 7.5.

18 mg BSA(MH)—BPRU was added and the mixture was stirred for a further60 min at a protein concentration of 9 mg/ml (pH 7.1; 25° C.). In orderto stop the reaction cysteine was added to a final concentration of 2mmol/l and stirred for a further 30 min at pH 7.1. N-methyl-maleimide(Sigma) was subsequently added to a final concentration of 5 mmol/l andit was stirred for a further 30 min at pH 7.1 and 25° C.

The mixture stopped in this manner was dialysed for 18 hours at roomtemperature (RT) against >500-fold volume 0.1 mol/Na-phosphate, 0.1%(w/v) SDS, pH 6.5 and purified over a Sephacryl S 200 column(Pharmacia). The most important general conditions for the columnoperation are: column volume 340 ml, application volume 12 ml, flowrate: 13.0 cm/hour, mobile buffer 0.1 mol/l Na-phosphate, 0.1% (w/v)SDS, pH 6.5, operating temperature RT.

The column operation was monitored at a wavelength of 280 nm by means ofa flow-through photometer and collected in fractions (fraction sizeabout 0.5% of the column volume).

After UV recording the fractions of the high molecular elution profilewere collected into a pool, the product was concentrated in an Amiconstirred cell with a YM30 membrane (Amicon) to a protein concentration of10 mg/ml and frozen at −80° C.

Incorporation: 5 mole p24 antigen per mole p24 antigen-BSA-BPRU. Yield:19 mg.

EXAMPLE 4

Improvement of the sensitivity of the bridge test format by usingmultimeric antigens

a) Carrier-Bound Multimeric Antigens (Polyhaptens)

Various variants of biotinylated polyhaptens were used in adouble-antigen immunoassay in combination with a monomericdigoxygenylated hapten and namely with the same molar amount ofbiotinylated or digoxigenylated hapten. The amino acid sequenceNNTRKSISIGPGRAFYT from the gp120 region of HIV was used as the epitope.The haptens were synthesized as described in examples 1 and 2. Therelative reactivity of native anti-HIV sera with the variousbiotinylated polyhaptens was standardized to the reactivity of sera withthe corresponding biotinylated monomeric hapten (=100% reactivity). Theresults of this experiment are shown in Table 4. Effective loadingReactivity: per carrier compared to molecule monomeric antigen Carriermolecule biotin peptide (= 100%) BSA (MW: 69000) 1 4.2 ca. 173.0% (ca.627 Aa) 1 5.1 ca. 185.0% β-Gal (MW: 465000) 1 2.2 ca. 123.5% (ca. 4227Aa) 1 3.6 ca. 151.8% 1 9.4 ca. 125.0% Bovine-Fab (MW: 75000) 1 5.9 ca.146.0% (ca. 682 Aa)b) Multimeric Branched Antigens

Biotinylated and ruthenylated antigens with monomeric or multimericbranched epitopes were compared in a double antigen immunoassay in abridge test format.

In the case of one epitope from the NS4 region of HCV (sequenceSRGNHVSPTHYVPESDAA) the combination of a monomeric biotinylated antigenand a monomeric ruthenylated antigen was compared with the combinationof multimeric branched biotinylated antigen (see Table 3d, line 2) and amonomeric ruthenylated antigen in a bridge test. The signaldifferentiation was determined i.e. the ratio in the measured signalbetween positive and negative samples. A higher signal differentiationmeans a better sensitivity. When using a multimeric biotinylated antigena signal differentiation of 386 compared to a signal differentiation ofonly 208 for the combination of both monomeric antigens was obtained.

A double antigen bridge test was carried out correspondingly using anantigen sequence from the gp120 region of HIV. The epitope used has theamino acid sequence NTTRSISIGPGRAFY. A combination of a monomericbiotinylated and a monomeric ruthenylated antigen was compared with acombination of a multimeric branched biotinylated antigen (see Table 2d,line 2) and a multimeric ruthenylated antigen (see Table 2d, line 4). Ina test using the combination of the two multimeric antigens a signaldifferentiation between a positive and negative sample of 12 was found.In contrast the combination of both monomeric antigens only has a signaldifferentiation of 10.

EXAMPLE 5

Improvement of the Sensitivity of the Bridge Test Format by UsingMultimeric Carrier-Bound Antigens

A combination of a monomeric biotinylated and a monomeric ruthenylatedantigen, was examined in a bridge test together with a combination of amonomeric biotinylated antigen and a carrier-bound multimericruthenylated antigen (carrier molecule: bovine serum albumin; epitope:HIV-p24 antigen; produced according to example 3) and a combination ofcarrier-bound multimeric biotinylated antigen and a monomericruthenylated antigen. In two different positive samples (HIV sera) asignal differentiation positive/negative of 2 was found in each casewith the combination of the two monomeric antigens whereas thecombination of a monomeric biotinylated antigen and a multimericruthenylated antigen yielded a differentiation of 19 and 7 and thecombination of the multimeric biotinylated antigen and a monomericruthenylated antigen yielded a differentiation of 4 and 3.

Even when using a combination of a monomeric biotinylated antigen andanother multimeric ruthenylated antigen (carrier molecule: rabbitimmunoglobulin) a much larger signal differentiation ofpositive/negative of 3, 22 and 10 was found in three different positivesamples compared to 2, 9 and 8 for a combination of the monomericantigens.

Even when using another epitope (recombinant protein from the HIV-gp41region) it was possible to demonstrate the superiority of the multimericantigens compared to the monomeric antigens. Whereas in the case of acombination of monomeric biotinylated and digoxigenylated antigenspractically no differentiation between negative and positive was foundin the bridge test, the combination of multimeric polyhaptens showed avery good differentiation.

EXAMPLE 6

Improvement of the Sensitivity of the Bridge Test Format by UsingMultimeric Antigens

A combination of an immobilized monomeric antigen and a labelledmultimeric antigen is particularly preferred to achieve an optimalsensitivity over a broad concentration range of specific immunoglobulin.The preferred amounts used are 1 equivalent immobilized epitope to0.2-10 and in particular 0.2-8 equivalents labelled epitopes.

FIG. 2 shows a comparison of a combination of a monomeric biotinylatedantigen and a monomeric ruthenylated antigen in an epitope ratio of 1:1(curve 1) and a combination of a monomeric biotinylated antigen and amultimeric ruthenylated carrier-bound antigen in an epitope ratio of 1:2(curve 2) and 1:4 (curve 3).

The sequence stated in example 4a from the gp120 region of HIV was usedas the epitope. The carrier molecule for the multimeric antigen was BSA.The loading of the carrier with epitope groups was 5:1 and 3:1 with theBPRu groups, each on a molar basis.

It is apparent from FIG. 2 that the use of multimeric antigens leads toa reduction of the Hook effect and to a general increase in thesensitivity.

EXAMPLE 7

Improvement of the sensitivity of the bridge test format when usingmultimeric antigens by increasing the number of marker groups.

A further improvement of test sensitivity is achieved due to the factthat it is possible to increase the number of marker and solid phasebinding groups to a large extent without masking the epitope regions orincreasing the unspecific background values by increasing thehydrophobicity.

Digoxigenylated multimeric antigens were compared which containedepitopes from the gp120 region of HIV (cf example 4b) coupled to abovine Fab antibody fragment carrier. In each case the carrier wasloaded with the peptide epitope in the range of 1:6 to 1:7 on a molarbasis. The loading of the carrier with digoxigenin groups was 1:2 and1:4.

The results of this experiment are shown in Table 5. It can be seen thata non-linear improvement in sensitivity and a considerable reduction inthe Hook effect was achieved by increasing the number of marker groups.TABLE 5 Stoichiometry Stoichiometry carrier: Dig. carrier: Dig. Sample1:4 1:2 Dynamics of the measuring range Dilution steps mA mA 1/16384 36148 1/8192 48 159 1/4096 49 151 1/2048 82 158 1/1024 132 159 1/512 302157 1/256 675 164 1/128 1503 190 1/64 3493 259 1/32 7436 480 1/16 93061066 1/8 9449 3036 1/4 9449 3378 1/2 9449 2694 undiluted 9474 2266

EXAMPLE 8

Improvement of the Stability by Using Multimeric Antigens

The stability of monomeric and multimeric antigens was tested. For thispurpose the signal recovery after a three day incubation at 35° C.relative to the original signal intensity was determined.

A signal recovery of 3.0 and 4.0% for two samples was determined for amonomeric ruthenylated antigen from the gp120 region of HIV (sequencesee example 4) in combination with a fresh biotinylated monomericantigen. When using a carrier-bound multimeric ruthenylated antigen(carrier: rabbit IgG, 4 marker groups and 3 epitopes per carriermolecule) a signal recovery of 73.1 and 73.6% was determined under thesame test conditions.

A biotinylated monomeric antigen with the same epitope sequence wasexamined in a similar manner together with a carrier-bound biotinylatedmultimeric antigen (carrier: rabbit IgG, 18 biotin groups and 3 epitopesper carrier) in combination with a monomeric ruthenylated antigen.Signal recoveries of 25.0 and 37.0% were determined for the monomericbiotinylated antigen and 120.3 and 79.9% for the multimeric antigen.

EXAMPLE 9

Improvement of the Sensitivity with Respect to the Reactivity withAntigens of Low Affinity

Multimeric antigens are preferably used to detect specificimmunoglobulins of low affinity e.g. in the case of a recentseroconversion and in the case of new viral subtypes.

a) Ruthenylated Multimeric Antigens

The positive/negative signal differentiation was examined using antigenswith an epitope sequence from the NS4/3 region of HCV. A combination ofa monomeric ruthenylated and a monomeric biotinylated antigen resultedin a positive/negative signal differentiation of 3 and 1 in twodifferent positive seroconversion samples i.e. a positive sample was notrecognized as such. When multimeric IgG carrier-bound biotinylated andruthenylated antigens were used a signal differentiation of 21 wasdetermined in each case. Only the use of multimeric antigens enables thepositive samples to be correctly classified.

b) Biotinylated Multimeric Antigens

The same peptide epitope from the gp41 region of HIV (gp41/3) wascompared in each case as a carrier-bound multimeric antigen and as amonomeric antigen. In each case 50 ng/ml biotinylated anddigoxigenylated monomeric peptide was used. In the case of themultimeric antigens 50 ng/ml “peptide equivalent” was used in which theamount of peptide was calculated from the degree of loading of thepolyhapten. The test was carried out on an ES700 automated analyzer.

The test was carried out using various seroconversion panels as samples.FIG. 3 shows that the panels were correctly classified as positive intests using the digoxigenylated polyhapten whereas a false negativeresult was obtained when using the monomeric antigen. The cut-off indexis the boundary between negative and positive evaluation of anexperiment. It is defined as the double value of the negative control.

1. A method for detection of an antibody against a pathogenic organismin a liquid sample, wherein said pathogenic organism is selected fromthe group consisting of bacteria, viruses and protozoa, the methodcomprising a) incubating (1) said sample, (2) a solid phase, (3) a firstantigen for said antibody, wherein the first antigen has at least onemarker group, and (4) a second antigen for said antibody, wherein thesecond antigen binds to the solid phase, under conditions to obtain acomplex comprising a solid phase-bound second antigen to which is boundthe antibody and to which is bound the first antigen; and b) detectingsaid antibody by direct or indirect detection of the marker group; andwherein at least one of said antigen is of formula (la) or (lb)(P—)_(n)T(-L)_(m)  (Ia)T(—P-L_(m))_(n)  (lb) wherein T is a carrier, P is a peptide comprisingan epitope region wherein said epitope region is reactive with theantibody, L is the marker group or a group which binds to the solidphase, -is a covalent coupling, n is 240 and m is 1-10.
 2. The method ofclaim 1, wherein the first antigen comprises multiple epitope regions,said epitope regions being identical in amino acid sequence.
 3. Themethod of claim 1, wherein the second antigen comprises multiple epitoperegions, said epitope regions being identical in amino acid sequence. 4.The method of claim 1, wherein the first antigen and the second antigencomprise multiple epitope regions, said epitope regions being identicalin amino acid sequence.
 5. The method of claim 1, wherein the at leastone marker group comprises a metal chelate marker group.
 6. The methodof claim 1, wherein said indirect detection of said antibody comprises:c) providing in step b) the first antigen having the marker groupcomprising a hapten and a binding partner for the hapten being labeledwith a signal generating group; and d) detecting the antibody bydetecting the signal-generating group.
 7. The method of claim 6, whereinthe hapten is selected from the group consisting of a sterol, a bileacid, a sexual hormone, a corticoid, a cardenolide, acardenolide-glycoside, a bufadienol, a steroid-sapogenine and a steroidalkaloid, and wherein the specific binding partner comprises an antibodyfor the hapten.
 8. The method of claim 1, wherein the second antigen isbiotinylated and the solid phase is coated with streptavidin or avidin.9. The method of claim 1, wherein the at least one of the first antigenand the second antigen comprises a carrier to which the epitope regionsare covalently coupled, wherein the carrier is non-reactive with theantibody.
 10. The method of claim 9, wherein the carrier is a natural orsynthetic peptide or polypeptide or a synthetic polysaccharide.
 11. Themethod of claim 10, wherein the carrier is selected from the groupconsisting of an albumin, an immunoglobulin, an immunoglobulin fragment,a β-galactosidase, a polylysine and a dextran.
 12. The method of claim1, wherein P is a synthetic peptide sequence of 6 to 50 amino acids. 13.The method of claim 12, wherein the synthetic peptide sequence is amultimeric antigen comprising multiple, identical epitope regions and animmunologically inactive spacer region, said epitope regions beingidentical in amino acid sequence.
 14. The method of claim 1, wherein Pis a recombinant polypeptide sequence comprising a length of up to 1000amino acids, wherein the polypeptide sequence comprises a single epitoperegion or a multiple of an epitope region.
 15. The method of claim 1,wherein the first antigen and the second antigen is a recombinant fusionpolypeptide wherein P is a mosaic peptide comprising multiple,immunologically reactive epitope regions optionally linked byimmunologically inactive spacer regions.
 16. A reagent for detection ofan antibody against a pathogenic organism in a liquid sample, whereinsaid pathogenic organism is selected from the group consisting ofbacteria, viruses and protozoa, the reagent comprising 1) a solid phase;2) a first antigen for the antibody, wherein the first antigen has atleast one marker group; and 3) a second antigen for the antibody,wherein the second antigen binds to the solid phase, wherein at leastone of said antigen is of formula (Ia) or (Ib)(P—)_(n)T(-L)_(m)  (Ia)T(—P-L_(m))_(n)  (Ib) wherein T is a carrier, P is a peptide comprisingan epitope region, wherein said epitope region is reactive with theantibody, L is the marker group or a group which binds to the solidphase, -is a covalent coupling, wherein n is 240 and wherein m is 1-10.17. The reagent of claim 16, wherein the at least one marker groupcomprises a hapten and the reagent further comprises a specific bindingpartner for the hapten, wherein the specific binding partner has asignal-generating group.
 18. The reagent of claim 16, wherein the secondantigen comprises multiple, identical epitope regions and isbiotinylated, and wherein the solid phase is coated with streptavidin oravidin.
 19. The method of claim 1, wherein P comprises at least onebranching site of the formulaP¹{P²[P³(P⁴)_(t)]_(s)}_(r) wherein P¹ through P⁴ are each an amino acidsequence having a length of up to 50 amino acids wherein at least two ofP¹ through P⁴ comprise a copy of the single epitope and r is 1 or 2, sis an integer from 0 to 4 and t is an integer from 0 to 8, with theproviso that r, s and t are selected to result in P containing the atleast one branching site and the several copies of the single epitope.20. The method according to claim 19, wherein the at least one branchingsite is formed by a trifunctional amino acid.
 21. The method of claim 1,wherein the several copies of the single epitope are directly covalentlycoupled to each other or are indirectly bound to each other via spacerregions which are covalently coupled between the copies.
 22. The methodaccording to claim 20, wherein the at least one branching site is formedby lysine, ornithine or both.
 23. An antigen of the formula (1a) or (1b)(P—)_(n)T(-L)_(m)  (1a)T(—P-Lm)_(n) (1b) wherein T is a carrier, P is a peptide comprising anepitope region, wherein said epitope region is reactive with an antibodyto be determined, L is a marker group or a group which is bindable to asolid phase, -is a covalent coupling, n is 2-40 and m is 1-10.
 24. Anantigen having at least one marker group or group which is bindable to asolid phase, several copies of a single epitope which is reactive withan antibody to be determined and at least one branching site, and is ofthe formulaP¹{P²[P³(P⁴)_(t)]_(s)}_(r) wherein P¹ through P⁴ are each an amino acidsequence having a length of up to 50 amino acids wherein at least two ofP¹ through P⁴ comprise a copy of the single epitope and r is 1 or 2, sis an integer from 0 to 4 and t is an integer from 0 to 8, with theproviso that r, s and t are selected to result in an antigen containingthe at least one branching site and the several copies of the singleepitope.